Figure 2. The plot of the ladder displaying base pairs that correspond to the distance traveled on the 1% agarose gel by the HindIII digest of lambda phage DNA. When lambda phage DNA is digested with the restriction enzyme HindIII, it is cut between the two 'A' nucleotides producing sticky ends, which yields 8 fragments. In the lab there was the appearance of 5 of these 8 bands. The y-axis was logged to produce a straight line and the exponential equation was used to calculate the corresponding base pairs for the bands.
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The RFLP analysis was performed on DNA that was obtained from a large quantity of blood that was on the floor at the crime scene.
A double digest was performed on the blood collected and for the 4 suspects in the investigation. Size is inversely proportional to the distance traveled in the gel so therefore the shortest fragments will travel the furthest. The distance from the bottom of the well to the middle of the band was measured for analysis. The ladder that was used with the RFLP analysis was a HindIII digest of lambda phage DNA. |
From figure 1 the base pairs can be calculated for each of the subsequent bands that are present on the gel. The equation of the line in figure 1 can be used to calculate these values and and the ladder reference measurements can be seen in table 1.
Table 1. The HindIII digest of lambda phage DNA was used as the ladder and reference for the crime scene and suspect DNA and the measured distance of each of the bands and the corresponding base pairs that were calculated using the exponential equation obtained from the ladder base pairs. The band distance for the crime scene and each of the suspect's DNA was measured from the bottom of the wells to the middle of the bands. The values obtained were then used to calculate base pairs present in each band using the equation y=28874e^-0.115x, where x, was the distance migrated. The base pairs present in each band were used to identify if a suspect was present at the crime scene.
Distance (mm) |
Base pairs |
Suspect |
Distance Migrated (mm) |
Base Pairs (bp) |
11 |
9416 |
CS |
20.0 21.5 31.5 |
2894 2436 771 |
13 |
6557 |
A |
22.5 28.5 30.5 |
2171 1089 865 |
14.5 |
4361 |
B |
22.5 26.5 29.0 |
2171 1370 1028 |
22 |
2322 |
C |
20.0 21.5 31.5 |
2894 2436 771 |
23.5 |
2027 |
D |
22.5 30.0 36.0 |
2171 916 459 |
Sample Calculation: y=28874e^(-.0115x) where x is plugged in as the distance of another band to determine the base pairs.
y=28874e^(-0.115*20) y=2894 base pairs
From the RFLP analysis of the fresh blood at the crime scene is was determined that suspect C was present at the crime scene because their DNA was found there. The number of base pairs that were found for both the CS and suspect C data were: 2894, 2436 and 771 for each band on the gel respectively.
y=28874e^(-0.115*20) y=2894 base pairs
From the RFLP analysis of the fresh blood at the crime scene is was determined that suspect C was present at the crime scene because their DNA was found there. The number of base pairs that were found for both the CS and suspect C data were: 2894, 2436 and 771 for each band on the gel respectively.